The innate immune system is the first line of defense against invading microorganisms. When the innate immune system fails to be activated, lethal infection ensues. Conversely, the inappropriate activation of innate immunity can lead to a variety of illnesses, including sepsis, rheumatoid arthritis and lupus. Insects rely solely on an innate immune response to combat pathogens. The study of innate immunity in Drosophila, a genetically tractable organism for which advanced genetic methods are available, allows for the rapid progress in the study of innate immunity in the absence of the confounding influence of the adaptive immune response. The study of Drosophila immunity has resulted in important contributions to our understanding of immunity in humans, such as the identification of Toll receptors. The long-term goal of this proposal is to elucidate the molecular mechanisms responsible for the recognition of gram-negative bacteria and the subsequent activation of the signal transduction pathways which culminate in the expression of antimicrobial peptide genes in Drosophila. The IMD signal transduction pathway is critical for immune activation and survival following gram-negative infection. The proposed experiments will characterize the gram-negative bacterial products that are recognized by the receptor PGRP-LC, and uncover the mechanisms by which PGRP-LC activates the IMD intracellular signaling pathway. This pathway requires the kinases dTAK1 and Drosophila IKK as well as the caspase Dredd, and culminates in the activation, by caspase-mediate cleavage, of the Drosophila NF-kB homolog Relish. We propose to investigate the mechanism(s) by which the cleavage of Relish is controlled by IKK-mediated phosphorylation. We will characterize a parallel signal transduction pathway, also activated via PGRP-LC, that involves JNK kinas activation, and results in the expression of a number of novel genes likely to be involved in host defense. We believe that the successful completion of our Aims is likely to lead to a better understanding of the innate immune response, in humans and flies. Such an understanding is critical to our ability to devise more effective therapies against inflammatory disease.